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Abstract

Introduction

Chagas disease remains a major cause of mortality in several countries of
Latin America and has become a potential public health problem in
non-endemic countries as a result of migration flows. Cardiac involvement
represents the main cause of mortality, but its diagnosis is still based on
nonspecific criteria with poor sensitivity. Early identification of patients
with cardiac involvement is desirable, since early treatment may improve
prognosis. This study aimed to assess the role of diastolic dysfunction,
abnormal myocardial strain and elevated brain natriuretic peptide (BNP) in
the early identification of cardiac involvement in Chagas disease.

Conclusion/Significance

In conclusion, the combination of diastolic function and BNP measurement adds
important information that could help to better stratify patients with
Chagas disease.

Author Summary

Chagas disease remains a major cause of morbidity and mortality in several
countries of Latin America and has become a potential public health problem in
countries where the disease is not endemic as a result of migration flows.
Cardiac involvement represents the main cause of mortality, but its diagnosis is
still based on nonspecific criteria with poor sensitivity. Early identification
of patients with cardiac damage is desirable, since early treatment may improve
prognosis. Diastolic dysfunction and elevated brain natriuretic peptide levels
are present in different cardiomyopathies and in advanced phases of Chagas
disease. However, there are scarce data about the role of these parameters in
earlier forms of the disease. We conducted a study to assess the diastolic
function, regional systolic abnormalities and brain natriuretic peptide levels
in the different forms of Chagas disease. The main finding of our investigation
is that diastolic dysfunction occurs before any cardiac dilatation or motion
abnormality. In addition, BNP levels identify patients with diastolic
dysfunction and Chagas disease with high specificity. The results reported in
this study could help to early diagnose myocardial involvement and better
stratify patients with Chagas disease.

Introduction

Chagas disease, a major cause of morbidity and mortality in several countries of
Latin America [1], has become a potential public health problem in
countries where the disease is not endemic as a result of migration flows [2], [3], [4]. Chagas
cardiomyopathy is the most serious form of the chronic phase of the disease and
represents the major cause of mortality in these patients. For this reason, accurate
diagnosis of cardiac involvement is critical. However, Chagas disease remains a
neglected disease [5] and the diagnosis of Chagas cardiomyopathy is
still based on simple and nonspecific criteria including an increased cardiothoracic
ratio (>0.5) or ECG abnormalities such as complete right bundle-branch block,
left anterior hemiblock, complete left bundle-branch block, as well as other
conduction and rhythm disturbances [6], [7].
Echocardiography refined the diagnosis of Chagas cardiomyopathy, and regional wall
motion abnormalities, reduced left ventricular ejection fraction (LVEF)
<50% and increased left ventricular (LV) end-diastolic diameter
>55 mm are now included as diagnostic criteria in some publications [8],
[9]. In
spite of that, the sensitivity of these parameters is far from perfect and they may
indeed misclassify patients with early myocardial involvement into the undetermined
form, as conventional 2D echocardiography only detects advanced myocardial
involvement. On the other hand, patients without cardiac disease but having one of
the described as typical but unspecific ECG findings could be considered to have
Chagas cardiomyopathy. Therefore, a more accurate classification model, particularly
to identify patients with early cardiac involvement from the undetermined form would
be desirable, since an early treatment and closer follow-up might be beneficial on
these patients [8].

Analysis of diastolic function by echocardiography, cardiac magnetic resonance (CMR)
and several biomarkers, including brain natriuretic peptide (BNP) and inflammation
markers, have emerged as useful tools in the diagnosis and monitoring of heart
failure in different conditions. In fact, BNP has recently been included in the
guidelines for the diagnosis and management of congestive heart failure [10].
Comprehensive evaluation of diastolic dysfunction and myocardial strain imaging has
provided more accuracy and sensitivity to detect early myocardial involvement in
different cardiomyopathies [11], [12]. However, these
methods have been seldom utilized in characterizing patients with established Chagas
cardiomyopathy [13], [14], [15], and its role in the
identification of cardiac involvement in the earlier phases of the disease is
unclear [16], [17], [18].

We conducted a prospective study aimed to analyze the added value of different
techniques in identifying cardiac involvement in the undetermined stage of Chagas
disease. Diastolic function, natriuretic peptide and inflammatory markers levels in
different phases of the disease were measured and correlated with longitudinal and
radial myocardial strain and delayed enhancement on CMR to find out their value in
improving the stratification of patients with Chagas disease.

Methods

Study Population

A cross-sectional analysis was performed in a prospective cohort of consecutive
adult patients evaluated at our Institution from January 2008 to June 2009.
Diagnosis of Chagas disease was based on a clinical record compatible with the
epidemiology of the disease (individuals from endemic zones of Chagas disease)
and microbiologic diagnosis by any combination of at least two positive
commercial serological tests using different antigens: ELISA using a T.
cruzi lysate (Ortho-Clinical Diagnostics®), ELISA with
recombinant antigens (BioELISA Chagas®, Biokit S.A.) and indirect
immunofluorescence (Inmunofluor Chagas, Biocientifica®). In an attempt
to avoid factors that could have an effect on diastolic function, myocardial
strain, natriuretic peptides or inflammatory markers levels, patients with
severe non-cardiac diseases, prior diagnosis of heart disease from other
etiology (ischemic, hypertensive or alcoholic), hypertension, diabetes mellitus,
active infections by other causal agent or previous treatment with benznidazol
were excluded. All patients gave written consent for inclusion. The research
protocol was approved by the Ethics Committee of our institution.

Clinical examination, blood analysis including ions, creatinin, inflammatory
markers and natriuretic peptides levels as well as a comprehensive
2D-echocardiogram with diastolic function and myocardial strain analysis were
obtained from all patients. CMR studies were performed in an unselected sample
of patients with Chagas disease (Groups 1–3).

Biochemical measurements

Measurements of plasmatic levels of endothelin 1, tumor necrosis factor-α
(TNFα), interleukin 6 (IL-6), atrial natriuretic peptide (ANP) and BNP
levels was carried out through peripheral venous puncture, after a 30 minutes
rest, and quantified using commercially available kits. BNP levels were measured
using a fully automated two-site sandwich BNP immunoassay on an Advia Centaur
(Siemens Diagnostics, Zurich, Switzerland). Minimum sensitivity and upper limit
of normal values are 2 and 37 pg/ml respectively for the BNP assay. The
precision of this technique is 1.8–4.3%.

Imaging techniques

Echocardiographic studies were performed with a commercially available system
(VIVID 7, General Electrics; Milwaukee, WI). Images were digitally stored for
later off-line analysis with a commercial software package (EchoPac, General
Electrics; Milwaukee, WI). LV volumes and LVEF were calculated using the
modified Simpson rule (biplane method). Left atrium area was measured at the
end-ventricular systole excluding the confluences of the pulmonary veins and the
left atrium appendage. LV volumes and left atrium area were indexed to body
surface area. Analysis of diastolic function was performed evaluating the mitral
inflow pattern with pulsed Doppler: E and A waves and deceleration time of the E
wave (DT); the pulmonary vein flow and the mitral annulus velocities with
Doppler Tissue Imaging (Em and Am). Patients were classified according to
diastolic function patterns (normal, impaired relaxation or stage I,
pseudonormal or stage II and restrictive pattern or stage III) following current
recommendations [19]. The ratio of early diastolic mitral flow
velocity to early diastolic mitral annulus velocity (E/Em) was used as a
surrogate of LV filling pressures. LV segmental myocardial longitudinal
2D-strains were acquired from two and four-chamber apical views and radial
strains from the parasternal short axis view at the level of the papillary
muscles. Averages of longitudinal and radial strains were obtained by dividing
the sum of all segmental strains by the number of analyzed segments. Images were
optimized to obtain a frame rate >50 fps.

CMR studies were performed using a 1.5 T clinical scanner (Signa CV HDxt, General
Electric, Milwaukee WI). Functional assessment was studied with a standard cine
steady-state free precession sequence and delayed-enhanced images were acquired
using a gradient-echo segmented inversion recovery technique, 10 minutes after
intravenous administration of gadodiamide at a dose of 0.2 mmol/Kg (Omniscan, GE
Healthcare, Madrid). LV end-diastolic, end-systolic volumes and LVEF were
calculated using Mass 4.0.1 software analysis (MEDIS, The Netherlands). All
echocardiographic and CMR analyses were performed by experienced independent
observers blinded to BNP measurements and clinical data.

Statistical analysis

Continuous baseline variables were expressed as mean±SD or median
(interquartile range) values depending on normality assessed by the
Shapiro-Wilks test. Categorical variables were expressed as total number
(percentages) and compared between groups using Chi-square test or
Fisher's test. Differences in continuous variables were analyzed using
either ANOVA test or Kruskall Wallis test depending on variable distribution.
Post-hoc analysis using either T-test or Wilcoxon test corrected by Bonferroni
method was carried out to detect differences between each pair of groups. Trends
in continuous variable changes across Chagas's disease severity and
diastolic function impairment were analyzed using either ANOVA test for trend or
Jonckheere-Terpstra test. Correlations between natriuretic peptides and LV
volumes and LVEF were assessed using Pearson coefficient. Receiver-operating
characteristic (ROC) curves were constructed to estimate the accuracy of BNP and
ANP to detect any grade of diastolic dysfunction. Statistical analysis was
performed with SPSS 15.0®.

Results

A total of 98 consecutive subjects were included. Median age was 37 (range 20 to 58)
years and 40% were men. There were 44 subjects in Group 0, 32 patients in
Group 1, 14 in group 2, and 8 patients in group 3. Clinical characteristics,
hemodynamic data, and levels of inflammatory markers and natriuretic peptides are
shown in Table 1. Creatinin
and sodium plasmatic levels were normal for all patients. Only two patients were
under pharmacological treatment: one patient in group 1 was treated with
betablockers and one patient in group 3 was under angiotensin converting enzyme
inhibitors. There were no differences in clinical and hemodynamic characteristics
between groups except for the New York Heart Association (NYHA) functional class.
The majority of patients were in NYHA functional class I; no patient had NYHA
functional class III or IV. TNFα levels were higher in group 1 as compared
to group 0 and a significant trend towards increasing levels was observed according
worsening clinical forms of Chagas disease. Abnormally high BNP levels (>37
pg/ml) were noted in 0%, 13%, 29% and
63% of patients in groups 0, 1, 2 and 3, respectively. BNP and ANP levels
in group 3 were significantly elevated as compared to those in groups 0 and 1. There
were no statistically significant differences in IL6 and endothelin 1 levels between
groups. IL6 levels were undetectable in 68% of patients.

Echocardiographic parameters of control individuals and patients in the
undetermined and cardiac forms.

When patients were classified according to the diastolic function pattern,
50% of patients in groups 1 and 2 had an impaired relaxation pattern. On
contrast, every patient in group 3 had a certain degree of diastolic dysfunction
(Table 3). Two
(5%) subjects in group 0 had impaired relaxation pattern. Natriuretic
peptides and LV myocardial strain averages were progressively different as diastolic
dysfunction severity increased (p for trend <0.01) (Table 4). The accuracy of BNP and ANP to detect
any grade of diastolic dysfunction, as assessed by the area under the ROC curve, was
0.73, 95%CI 0,60–0.85 for BNP and 0.70; 95%CI 0.58
– 0.81 for ANP. In addition, both BNP and ANP significantly correlated
with LV end-diastolic volumes (r=0.35;
p=0.001 and
r=0.26;
p=0.013, respectively), LV end-systolic volumes
(r=0.44; p<0.001 and
r=0.36; p<0.001 respectively) and LVEF
(r=−0.44; p<0.001 and
r=−0.44; p<0.001, respectively).

Serum and myocardial strain according to diastolic function
classification in patients with Chagas disease.

When only patients in the undetermined form of the disease (group 1) were considered,
BNP levels were higher in patients with diastolic dysfunction compared to those with
normal diastolic pattern (27±26 versus 11±8 pg/ml,
respectively, p=0.03). A similar result was
obtained in group 2, thus, a trend towards increasing BNP levels was observed from
normal diastolic pattern to abnormal relaxation and pseudonormal pattern
(11±4, 37±36 and 41±3, pg/ml respectively,
p=0.06). The areas under the ROC curves for BNP
and ANP to detect mild diastolic dysfunction in patients in the undetermined form
were 0.69; 95%CI 0,49–0.89 for BNP and 0.62; 95%CI
0.43–0.82 for ANP. Additionally, every patient with abnormally high levels
of BNP (>37 pg/ml) had diastolic dysfunction (Figure 1).

Distribution of patients according to the conventional
2D-echocardiography, ECG and diastolic function and prevalence of pathologic
levels of BNP (% of patients).

A CMR was performed in 21 Chagas disease patients, 7 patients in each group (groups
1–3) according to the standard classification [20], [21]. Two
(28%) patients in group 1 (Figure 2), 1 (14%) patient in group 2 and 3 (43%)
patients in group 3 had gadolinium delayed enhancement compatible with scar or
fibrosis. However, when these patients were classified according to the diastolic
function pattern, none with normal diastolic function had delayed enhancement,
whereas 40% of patients with impaired relaxation pattern and
50% with pseudonormal pattern showed delayed enhancement (Figure 3).

Discussion

The main finding of our investigation is that diastolic dysfunction occurs before any
LV dilatation, regional or global systolic abnormalities or significant increased
filling pressures as assessed by E/Em ratio. In this regard, we found significant
reduction in Em and lengthening in DT in patients in the undetermined form compared
to control individuals, in spite of preserved LV volumes and systolic function. The
fact that all the rest of the echocardiographic measurements remained similar in
both the undetermined form and control individuals, suggests that Em and DT could be
the most sensitive echocardiographic parameters to detect cardiac involvement by
Chagas disease. In addition, BNP levels identify patients with diastolic dysfunction
among those in the undetermined form of Chagas disease with high specificity. The
study also confirmed the fact that as the disease progresses from the undetermined
form to Chagas cardiomyopathy with abnormal 2D-echocardiography, including
enlargement of LV volumes and a deterioration of global LV function, diastolic
function deteriorates. This is supported also by the fact that global LV strain,
both longitudinal and radial, also progressively decreases along with diastolic
function impairment, suggesting the existence of myocardial damage, despite
preserved LVEF.

Previous studies analyzing diastolic function in the undetermined form of Chagas
disease have shown conflicting results (15–21). Barros et al. were the
first to report early LV diastolic dysfunction in patients in the undetermined form
of Chagas disease as they demonstrated a lengthening of DT and isovolumic relaxation
time [16].
Cianciulli et al. similarly observed that transmitral Doppler flow allowed to
identify early abnormalities of diastolic function in patients with normal ECG and
conventional 2D-echocardiogram [17]. On the other hand, Pazin-Filho et al. also
focused on patients in the undetermined form and showed that patients with normal
global and segmental LV systolic function by 2D echocardiography did not show any
abnormality of diastolic function [18]. However, in a
meticulous evaluation of this small study a trend for higher LA volumes, DT
lengthening and Em reduction could be observed among groups, and therefore, an
insufficient statistical power might have contributed to the negative result.

Despite being only performed in a small subgroup of patients, the results of CMR also
sustain that diastolic dysfunction may be the first manifestation of myocardial
involvement, before systolic dysfunction occurs as it happens in other
cardiomyopathies. Therefore, the comprehensive analysis of diastolic dysfunction
could be more sensitive in terms of early diagnosis of myocardial involvement
compared to the standard classification as it was shown that some patients with
normal ECG and normal conventional 2D-echocardiography had myocardial fibrosis
detected by delayed enhancement, whereas no patient with normal diastolic function
had enhancement on CMR. A prior study showed that delayed enhancement in CMR can be
present in up to 20% of patients who are in the undetermined form of the
disease [22], suggesting that this technique has an extended value
for the diagnosis in early stages of cardiac involvement. However, CRM availability
is limited particularly in areas where Chagas disease is endemic. The association
between a normal diastolic function by echo and absence of fibrosis on CMR has to be
confirmed in larger series.

Nevertheless, although diastolic function analysis seems to have a high sensitivity,
it has to be acknowledged that the complexity of diastolic dysfunction measurements
may preclude its use in large populations, especially in underdeveloped geographical
areas. In this regard, the initial screening with BNP determination could be
helpful, especially with the use of simplified, point-of-care kits. In fact, every
patient with abnormally high BNP levels had diastolic dysfunction in our study
(Figure 1).

Natriuretic peptides have been shown to be involved in the pathogenesis of Chagas
disease in animal experiments [23], [24] and some clinical studies have reported that BNP
levels are increased in Chagas cardiomyopathy [25], [26], [27] and correlates with
prognosis [28]. Ribeiro et al. demonstrated a high specificity
with moderate sensitivity for BNP to detect LVEF≤40% in infected
patients with an abnormal ECG or chest X-ray [25]. In a second study,
the same group reported that BNP levels correlated with LV dimensions and LVEF in
patients with Chagas disease and also that patients with mild degree of cardiac
dysfunction, defined as no more that minor alterations in their echocardiography,
had intermediate BNP levels compared to control individuals and patients in the
cardiac form of the disease [27]. In a third study, they compared the diagnostic
accuracy of the combination of BNP plasmatic levels and ECG vs. the standard
strategy (ECG and chest X-ray) to detect LVEF ≤40% and
demonstrated a significant improvement in specificity although the new strategy had
less sensitivity [26]. However, even though the correlation between BNP
levels and systolic dysfunction in Chagas disease has been well described, few
studies have correlated BNP levels and diastolic function in Chagas disease [14], [29];
indeed, all of them have been done in patients in the cardiac form of Chagas
disease. Barbosa et al [14] evaluated 59 patients with dilated cardiomyopathy
due to Chagas disease and reported a marked elevated concentration of the
amino-terminal portion proBNP specifically in patients with a restrictive diastolic
pattern. Oliveira et al [29] evaluated 36 patients, all of them with diffuse
or segmental ventricular motion abnormalities, and described a significant
correlation between BNP and E/E' ratio in the inferior wall. To our
knowledge there are no studies that have specifically assessed the association
between BNP and diastolic function in patients in the undetermined form. In our
population, the accuracy of BNP to detect any degree of diastolic dysfunction was
good (area under curve of 0.73); additionally, the ability to detect mild diastolic
dysfunction in the group of patients in the undetermined form was also good (area
under the curve of 0.69). The specificity of BNP levels >37 pg/ml to detect
mild diastolic dysfunction in patients in the undetermined form of Chagas disease
was 100%. The power of ANP levels to detect diastolic dysfunction was
slightly inferior”.

Different from ANP, BNP is mainly secreted in the ventricles in response to wall
stress, ischemia or fibrosis. Indeed, myocardial fibrosis has been described to
strongly trigger BNP synthesis [30]. Chagas cardiomypathy is a predominately
fibrogenetic cardiomyopathy. Cardiac fibrosis is evident even in early stages of the
disease [22], [31]. This fact might explain why BNP levels could be
elevated even in patients with normal NYHA functional class, normal LVEF and
ventricular filling pressures, and supports the idea that BNP levels measurement
could be useful to early detect cardiac involvement in Chagas disease. Enhanced
fibrosis, compared with other cardiomyopathies, could also explained higher BNP
levels in patients with Chagas disease as compared to patients with cardiac disease
of different etiologies in the same NYHA functional class [32].

Our study also aimed to assess the plasmatic levels of TNFα, IL6 and
endothelin 1, in patients with different clinical forms of Chagas cardiomyopathy.
These biomarkers have been described to be elevated in Chagas cardiomyopathy [33],
[34], [35]. We found statistically significant elevated
levels of TNFα in patients in the undetermined form as compared to control
individuals and a significant trend towards increasing levels was observed along
clinical severity groups. Our finding is in concordance with previously published
literature suggesting heart inflammation in patients with Chagas disease even in the
absence of heart failure. Talvani et al [35] had previously
reported higher TNFα levels in patients with severe Chagas cardimyopathy; in
this study although TNFα levels were slightly higher in patients in the
undetermined form as compared to those in healthy individuals, no statistically
significant differences were reached. Similarly, in our study, a trend towards
greater IL6 levels could be also observed along with progressive clinical severity
and diastolic function impairment. However, as this measurement was undetectable in
a significant proportion of patients, its interpretation is limited. The use of
high-sensitivity kits for IL6 detection could be valuable in this context. Finally,
our study failed to demonstrate differences in plasmatic levels of endothelin 1 in
the different forms of Chagas disease. A privious study reported elevated endothelin
plasmatic levels in patients with Chagas cardiomyopathy [33]. However, in this
study seropositive patients had similar endothelin plasmatic levels than control
individuals, and the group of patients in the undetermined form had even lower
levels than controls. Therefore, the usefulness of plasmatic measurement of
endothelin is not clear and more studies are required to clarify its role in the
clinical evaluation of patients with Chagas disease.

The main limitation of our study is its cross-sectional design and, consequently
progression of cardiomyopathy could not be evaluated. Longitudinal studies in Chagas
disease are difficult due to the slow progression of the disease and the frequent
change of residency that particularly patients who live in non-endemic areas have,
making follow-up difficult.

In conclusion, the initial screening with measurement of BNP, an easy test with high
specificity for LV damage, combined with a comprehensive analysis of diastolic
function that contributes with high sensitivity seems to be a more accurate strategy
to early diagnose LV involvement in Chagas disease. Our findings could help to
better stratify patients with Chagas disease. The association between normal
diastolic function, normal BNP levels, absent fibrosis on CMR and no progression of
the disease warrants confirmation in larger and prospective longitudinal studies.
Also, new studies are required to demonstrate that early treatment and closer
follow-up slow the disease progression and consequently improve the prognosis in
these patients.

Supporting Information

Checklist S1

Footnotes

The authors have declared that no competing interests exist.

This study was supported by grants from the Fondo de Investigaciones Sanitarias
(FIS), Instituto de Salud Carlos III, Madrid, Spain (PI 070773) and Red HERACLES
(RD06/0009/0008), http://www.isciii.es/htdocs/investigacion/Fondo_lineas.jsp. The
funders had no role in study design, data collection and analysis, decision to
publish, or preparation of the manuscript.

10. Hunt SA, Abraham WT, Chin MH, Feldman AM, Francis GS, et al. 2009 focused update incorporated into the ACC/AHA 2005 Guidelines
for the Diagnosis and Management of Heart Failure in Adults: a report of the
American College of Cardiology Foundation/American Heart Association Task
Force on Practice Guidelines: developed in collaboration with the
International Society for Heart and Lung Transplantation. Circulation. 2009;119:e391–479.[PubMed]